Advances in Bioanalysis as It Relates to ADME

Abstract

Bioanalysis continues to play a crucial role in ADME sciences. Most in vitro or in vivo studies have either a quantitative or qualitative bioanalytical endpoint. This chapter provides a general overview of the use of mass spectrometry in ADME sciences and the advantages and disadvantages of the various types of instruments.

Contents

8.1 Abbreviations 145

8.2 Basic Concepts 146

8.3 Sample Collection 147

8.4 Sample Extraction 147

8.5 Chromatographic Separation 147

8.6 Bioanalysis by LC-MS 149

8.7 Applications 157

References 162

Additional Readings 163

8.1 ABBREVIATIONS

ADME

Absorption, distribution, metabolism, and excretion

APCI

Atmospheric pressure chemical ionization

API

Atmospheric pressure ionization

APPI

Atmospheric pressure photo ionization

DART

Direct analysis in real time

DBS

Dried blood spot

DESI

Desorption electrospray ionization

ESI

Electrospray ionization

GC

Gas chromatography

GLP

Good laboratory practice

S.C. Khojasteh et al., Drug Metabolism and Pharmacokinetics 145 Quick Guide, DOI 10.1007/978-1-4419-5629-3_8, © Springer Science+Business Media, LLC 2011

HILIC

Hydrophilic interaction liquid chromatography

HPLC

High performance liquid chromatography

LC

Liquid chromatography

MALDI

Matrix-assisted laser desorption/ionization

MIM

Multiple ion monitoring

MRM

Multiple reaction monitoring

MS

Mass spectroscopy

P450

Cytochrome P450

PD

Pharmacodynamic

PK

Pharmacokinetic

SIM

Selected ion monitoring

SRM

Selected reaction monitoring

UHPLC

Ultra high performance liquid chromatography or ultra

high pressure liquid chromatography

UPLC

Ultra performance liquid chromatography

UV

Ultraviolet

8.2 BASIC CONCEPTS

Advances in bioanalytical sciences have played a critical role in improved integration of ADME (absorption, distribution, metabolism and excretion) sciences in drug discovery and development. Prior to the availability of LC-MS (liquid chromatography-mass spectrometry) techniques, most bioanalyses involved UV (ultra violet) analysis. The limited selectivity and sensitivity of UV analysis necessitated extensive sample clean up and long chromato-graphic gradients to increase selectivity. Therefore, most analyses were limited to determination of drug levels in support of toxicology and clinical studies. The introduction of LC-MS in the early 1990s allowed routine drug determination of drug levels in preclinical PK (pharmacokinetic) and efficacy studies as well in in vitro ADME studies.

Bioanalysis can be subdivided in to five sequential steps:

• Sample collection

• Sample extraction

• Chromatographic separation

• Bioanalysis by LC-MS

- Ionization

- One or more stages of mass analysis

- Fragmentation (if MS/MS is employed)

- Detection

• Data processing (not discussed here)

8.3 SAMPLE COLLECTION

For in vivo studies, blood is collected at specific timepoints. Reliable automated blood sampling devices are available for rodent studies. However, blood sampling for dog and monkey PK studies is still manual. Frequently, the blood is centrifuged to obtain plasma for analysis. In vitro ADME studies, such as P450 (cytochrome P450) inhibition or metabolic stability in microsomes or hepatocytes, are frequently performed using automated assays involving liquid handlers, and samples are collected at specific timepoints.

8.4 SAMPLE EXTRACTION

Because of the selectivity and sensitivity of LC-MS equipment, sample extraction from plasma and from samples of in vitro studies is usually limited to protein precipitation using 96-well or 384-well plates. If a low detection limit is required or if interference from an endogenous component occurs, more selective extraction procedures, such as liquid extraction and solid-phase extraction, may be required. Detailed steps of the sample extraction process are outlined in Table 8.1.

8.5 CHROMATOGRAPHIC SEPARATION

Reversed-phase high performance liquid chromatography (HPLC) is involved in most separation. The most common types of bonded phases are listed in Table 8.2. The analyte is retained by the bonded phase on the HPLC column and is eluted off the column using isocratic conditions or a gradient. With isocratic conditions, the % organic solvent and % water in the eluent is constant, which usually results in relatively broad signals due to limited chromatographic separation. When a gradient is employed, the % organic solvent is gradually increased over time to elute the analyte off the column, and chromatographic separation is improved. The availability of columns with particles less than 2 mm in diameter has improved chro-matographic separation, but they do result in an increased column back pressure, and high pressure pumps are required. This technology is referred to as ultra performance liquid chromatography (UPLC) or ultra high performance/pressure liquid chromatography (UHPLC), and it offers significant advantages because it reduces the gradient time significantly without a loss in chromatographic separation (Plumb et al. 2008). An interesting alternative is the use of fused core particles. These particles have a solid core (about 1.7 mm

Table 8.1. Detail of plasma sample extraction via protein precipitation, liquid-liquid extraction and solid phase extraction

Step

Plasma protein precipitation

Liquid-liquid extraction

Solid phase extraction

10 ll

Plasma Add organic solvent (e.g., methanol or acetonitrile) containing the internal standard Vortex

Centrifuge

Transfer supernatant Bioanalysis

Plasma Add internal standard

Plasma Add internal standard

Add immiscible organic solvent (e.g., ethyl acetate) Vortex

Transfer organic layer Evaporate organic solvent

Reconstitute residue in small volume of solvent compatible with bioanalysis Vortex Bioanalysis

Condition solid phase extraction cartridges or 96-well plate with solvents Load plasma sample on top of the sorbent Remove liquid using vacuum Wash sorbent with additional volumes of water or other appropriate solvent Elute analyte off the sorbent with a strong organic solvent

Evaporate organic solvent Reconstitute residue in small volume of solvent compatible with bioanalysis Vortex Bioanalysis

in diameter) surrounded by a layer of porous silica (about 0.5 mm thick). This technology offers similar chromatographic efficiency to UPLC, but does not require high pressure pumps.

Table 8.2. Most commonly employed HPLC column bonded phase types

Phase type

Examples

Alkyl Aryl

Alkyl or aryl cyano Polar-embedded phases

Fluorinated phases

C4, C8, C18, C30 chains Phenyl

C8/18 carbamate, C8/18 amide, C8/18 sulfonamide, other polar groups Fluoroalkyl, fluorophenyl

Hydrophilic interaction liquid chromatography (HILIC) is a special type of normal phase chromatography and is an alternative to regular reversed-phase chromatography for retention and separation of highly polar analytes. The stationary phase is quite polar, which fuels retention of the polar analytes. The analytes are eluted by gradually increasing the % water in the mobile phase, and the analytes elute in order of increasing hydrophilicity, which is the exact opposite of reversed-phase chromatography.

8.6 BIOANALYSIS BY LC-MS

The four integral steps to bioanalysis by mass spectrometry are:

• Ionization

• Mass analysis

• Fragmentation (if MS/MS is employed)

8.6.1 Ionization

The first step in bioanalysis is evaporation of the solvent and ionization of the analytes. The two most common ionization techniques are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Both ESI and APCI involve atmospheric pressure ionization (API).

8.6.1.1 Electrospray Ionization

In ESI, the HPLC column effluent elutes from a capillary carrying a high positive or negative voltage. This results in a Taylor cone as illustrated in Fig. 8.1 and the formation of small droplets with an excess positive or negative charge. A parallel and/or counter current of heated drying gas results in evaporation of solvent from the droplets, and the droplets become enriched in protonated ([M + H]+ ions) or deprotonated ([M - H]- ions) analyte ions. The subsequent steps are still the topic of debate. One possibility is that ionized analyte molecules are expelled from the charged droplets because of Coulombic repulsion. Alternatively, the droplets fragment into smaller droplets because of Coulombic repulsion, and this process proceeds until a single ionized analyte molecule is left.

ESI allows ionization of biomolecules such as proteins and oligonucleotides, and the resultant ions carry multiple charges (e.g., [M + nH]n+), which reduces the mass-to-charge ratio. Small molecules usually carry a single charge.

Drying gas

Drying gas

Nebulizer gas

Nebulizer gas

Vacuum

[M + H]+ ions to mass spectrometer

Figure 8.1. Electrospray ionization process in the positive ion mode.

Vacuum

[M + H]+ ions to mass spectrometer

Figure 8.1. Electrospray ionization process in the positive ion mode.

8.6.1.2 Atmospheric Pressure Chemical Ionization In APCI, the column effluent is very rapidly evaporated, and a discharge needle nearby generates a "cloud" of reagent ions formed from the solvent, which can transfer a positive or negative charge to the analyte of interest via proton transfer.

The question of whether to use APCI or ESI for quantitative bioanalysis has been debated at length, and some strengths and weaknesses of ESI and APCI are listed in Table 8.3. Nevertheless, both ionization techniques have been incorporated successfully in bioanalysis of small molecule drugs.

Table 8.3. Strengths and weaknesses of electrospray ionization and atmospheric pressure chemical ionization

ESI

APCI

Advantages

Compatible with liquid

Compatible with liquid

chromatography

chromatography

Ionization of small and

Ionization of small non-

large relatively polar

polar or moderately polar

molecules

molecules

Disadvantages

Prone to ion suppression

Thermal degradation of

effects, which reduces

temperature-sensitive

sensitivity and

analytes

reproducibility

Reduced ionization of

moderately polar

analytes

In contrast to older ionization techniques, such as electron impact ionization, both ESI and APCI are compatible with liquid chromatography and are considered mild, i.e., very little fragmentation occurs in the ion source. Thus, the protonated [M + H]+ ions or deprotonated [M — H]- and are transferred intact into the vacuum of the mass spectrometer for analysis. If the column effluent contains a significant amount of sodium or potassium, [M + Na]+ and/or [M + K]+ ions can be detected as well.

Other ionization techniques, such as atmospheric pressure photo ionization (APPI), desorption electrospray ionization (DESI), and direct analysis in real time (DART), are available as well, but not in common use. GC (gas chromatography)-MS combined with electron impact ionization is still preferred for analysis of volatile analytes.

8.6.2 Mass Analysis and Fragmentation

Mass spectrometers separate ions according to their mass-to-charge, m/z, ratio. Small molecules usually carry a single charge, and, therefore, their mass and mass-to-charge ratio are equivalent. Subsequent sections refer predominantly to the behavior of small molecules carrying a single charge. Multiple types of mass spectrometers are available, and each type has unique advantages and disadvantages.

Operation of a mass spectrometer in the MS mode allows separation of all ionized material according to mass. This provides a third dimension to the selectivity of the assay beyond sample extraction and chromatographic separation. However, the matrix can contain endogenous isobaric interference (i.e., ions with the same m/z ratio as the analyte). It is possible to address this by changing the sample extraction procedure or chromatographic separation, but it is easier to resolve this by switching to MS/MS analysis. The analyte of interest is selected with the first mass analyzer and fragmented by collision with an inert gas (e.g., helium, nitrogen, or argon). The second mass analyzer separates all fragment ions according to their mass-to-charge ratio. In the MS/MS mode, either a full scan spectrum can be acquired for metabolite identification or a specific fragment ion can be monitored for quantitative bioanalysis. The latter is referred to as selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) if multiple transitions (e.g., analyte + internal standard or analyte + metabolite(s) + internal standard) are monitored. In SRM mode, it is much less likely that an endogenous interference has the same m/z value as the analyte as well as the same m/z value for the fragment ion that is monitored. This fourth dimension in selectivity has enabled much shorter cycle times (2 min or less per sample) and, therefore, increased throughput. With ion trap instruments it is possible to consecutively isolate and fragment ions, thereby allowing MSn data to be generated. The advantages and disadvantages of MS and MS/MS are summarized in Table 8.4.

Thus, the selectivity of a reliable and sensitive quantitative LC-MS/MS assay is governed by the following parameters:

• Sample extraction

• Chromatographic separation

• First stage of mass selection

• Collision-induced fragmentation of ions and a second stage of mass selection

Table 8.4. Advantages and disadvantages of analysis in the MS and MS/MS modes

MS mode

MS/MS mode

Advantages

Disadvantages

Ease of use

Decreased selectivity and increased likelihood of interference; a long chromatography column may be required

Increased selectivity and decreased likelihood of interference; a short chromatography column is usually sufficient Tuning of mass spectrometer requires more time and expertise

The instrument is more expensive

8.6.2.2 Single Quadrupole Mass Spectrometers

In single quadrupole mass spectrometers, the ions travel between four rods that carry a combination of AC and DC voltages, which results in ions with a specific m/z ratio reaching the detector while others are deflected away. A complete mass spectrum can be obtained by scanning the quadrupole. Alternatively, a particular m/z ratio may be monitored, and this is called selected ion monitoring (SIM). It is possible to monitor multiple m/z ratios sequentially (i.e., multiple ion monitoring (MIM)) to detect one or more analytes and the internal standard.

8.6.2.3 Triple Quadrupole Mass Spectrometers

Triple quadrupole mass spectrometers are the most widely used instruments for quantitative bioanalysis. A triple quadrupole mass spectrometer is comprised of two sets of quadrupoles separated by a collision cell. The ions selected by the first quadrupole are fragmented into structured characteristic fragment ions, and these are separated in the second quadrupole according to their mass-to-charge ratio. Triple quadrupole mass spectrometers are also quite powerful for metabolite identification because of their ability to perform unique scan modes. The principles of constant neutral loss scanning and precursor ion mode scanning are illustrated in Fig. 8.2. The advantages and disadvantages of single and triple quadrupole mass spectrometers are summarized in Table 8.5.

Triple Quadrupole Scan Modes

Ionization

Fragmentation

Detection

ABC+

—»

A+ + BC

—>

A+

Product ion

AB + C+

C+

spectrum

ABD+

A+ + BD

A+

AB + D+

>

D+

ABC+

A+ + BC

A+

Precursor ion

AB + C+

spectrum

ABD+

A+ + BD

A+

AB + D+

ABC+

A+ + BC

Constant neutral

AB + C+

>

C+

loss spectrum

ABD+

A+ + BD

AB + D+

D+

Figure 8.2. Product ion, precursor ion, and constant neutral loss MS/MS modes feasible with a triple quadrupole mass spectrometer.

Table 8.5. Advantages and disadvantages of analysis in single and triple quadrupole mass spectrometers

Single quadrupole mass spectrometers

Triple quadrupole mass spectrometers

Advantages

Ease of use Very sensitive in SiM mode

Disadvantages

Unit mass resolution Limited full scan sensitivity Limited selectivity, which can affect sensitivity

Ease of use Very sensitive in SRM mode increased selectivity Constant neutral loss and precursor ion scanning available for metabolite identification Unit mass resolution Limited full scan sensitivity

8.6.2.4 Three-Dimensional and Linear Ion Trap Mass Spectrometers

Three-dimensional and linear ion traps operate by using a combination of AC and DC voltages to trap ions. A mass spectrum can be obtained by destabilizing their path inside the trap and ejecting them out of the trap toward the detector. The ability to consecutively isolate and fragment ions enables acquisition of

MSn spectra. This makes these instruments quite useful for metabolite identification if the exact structure of fragment ions is in doubt. However, ion traps are less commonly used for quantitative bioanalysis because their inability to perform SIM and SRM makes them less sensitive than single and triple quadrupole mass spectrometers.

8.6.2.5 Time-of-Flight Mass Spectrometers

The operation of time-of-flight mass spectrometers is based on the principle that all ions receive the same amount of kinetic energy upon entering the mass spectrometer (provided they all carry the same number of charges), and their velocity is a function of their mass. Since the length of the flight tube is fixed, low mass ions have a high velocity and a short flight time, whereas high mass ions have a low velocity and a long flight time. The mass resolving power is such that it is possible to measure the exact mass of ions (within 5-10 ppm of the calculated mass) instead of the nominal mass (with unit mass resolution). Table 8.6 provides the exact mass and isotopic abundance of atoms commonly found in drug molecules. If the exact mass of an unknown entity is available, it is feasible to narrow down the number of possible molecular formulas, which makes this technique powerful for metabolite identification. Frequently, the time-of-flight section is preceded by a quadrupole and a collision cell to enable MS/MS capabilities.

Table 8.6. Exact mass and abundance of specific isotopes of atoms commonly found in drug molecules

Atom

Exact mass

Isotope abundance (%)

1H

1.0078

99.985

12C

12.0000

98.9

13C

13.0034

1.1

14n

14.0031

99.6

16O

15.9949

99.8

19f

18.9984

100

32S

31.9721

95.0

33S

32.9715

0.8

34S

33.9679

4.2

35Cl

34.9689

75.5

37Cl

36.9659

24.5

79Br

78.9183

50.5

81Br

80.9163

49.5

8.6.2.6 Fourier Transform and Orbitrap Mass Spectrometers Fourier transform mass spectrometers use a cryogenically cooled superconducting magnet to trap ions. The ions circulate in a cell in the bore of the magnet with a frequency that characterizes its mass. By monitoring the frequency of the complex circular motion it is possible to obtain the exact mass of the analyte (within 5 ppm of the calculated mass). The three-dimensional nature of the device allows for acquisition of MSn spectra.

The orbitrap mass spectrometer was introduced about 5 years ago. An orbitrap is comprised of two circular electrodes with a small space in between. The electrostatic forces on the ions are balanced by the centrifugal forces on the ions resulting in a stable circular motion of the ions. Like the Fourier transform mass spectrometer, the orbitrap allows accurate mass measurements, but is easier to operate. Currently, the orbitrap is preceded by an ion trap, thus enabling acquisition of MSn spectra. The advantages and disadvantages of three-dimensional, linear ion trap, time-of-flight, Fourier transform, and orbitrap mass spectrometers are summarized in Table 8.7.

Table 8.7. Advantages and disadvantages of analysis in three-dimensional, linear ion trap, time-of-flight, Fourier-transform, and orbitrap mass spectrometers

Three-

dimensional

Fourier

and linear ion

Time-of-flight

transform and

trap mass

mass

orbitrap mass

spectrometers

spectrometers

spectrometers

Advantages

Ease of use

Accurate mass

Accurate mass

determination

determination

(within 5-10

(within 5 ppm)

ppm)

MSn capabilities

Increased

MSn capabilities

for metabolite

sensitivity in full

for metabolite

identification

scan MS and

identification

MS/MS modes

due to increased

duty cycle

Compact

Relatively cheap

Disadvantages

Low sensitivity

Low sensitivity

Low sensitivity

for drug

for drug

for drug

quantitation

quantitation

quantitation

(continued)

(continued)

Table 8.7 (continued)

Three-

dimensional

Fourier

and linear ion

Time-of-flight

transform and

trap mass

mass

orbitrap mass

spectrometers

spectrometers

spectrometers

(SIM and SRM

(SIM and SRM

(SIM and SRM

not feasible)

not feasible)

not feasible)

Constant neutral

Constant neutral

Constant neutral

loss and

loss and

loss and

precursor ion

precursor ion

precursor ion

scanning not

scanning not

scanning not

possible for

possible for

possible for

metabolite

metabolite

metabolite

identification

identification

identification

Limited full scan

Difficult to operate

Difficult to

sensitivity

operate

Unit mass

Very expensive

resolution

8.7.1 Quantitative In Vitro ADME Studies

Quantitative bioanalysis is the cornerstone of high capacity acquisition of in vitro ADME data, and triple quadrupole mass spectrometers are most commonly used for this purpose. The following in vitro ADME assays have been integrated in the screening cascades for projects in early as well as late drug discovery:

• Metabolic stability in microsomes, S9, hepatocytes or recombinant P450 enzymes

• Competitive P450 inhibition

• Mechanism-based or time-dependent P450 inhibition

• Plasma protein binding or drug binding in other matrices such as microsomes or brain

• Blood to plasma partitioning

• Permeability in Caco-2 or MDCK cells or PAMPA

• Efflux in MDCK or LLCPK cells that overexpress transporters such as p-glycoprotein and BCRP

Most studies do not involve preparation of a standard curve to determine the absolute concentration. Quantitation is usually relative to a t = 0 min sample, a -NADPH sample, or another control sample. The order and capacity in which these assays are integrated in screening cascades is issue driven and, therefore, varies from project to project. However, metabolic stability and competitive P450 inhibition are frequently tier one assays and are preferably performed in parallel with potency assays. To increase the capacity of these in vitro ADME assays, the following techniques can be deployed:

• Column switching or multiplexing using multiple parallel chro-matography columns connected to a single mass spectrometer (see Fig. 8.3) ensures that the column effluent is monitored only around the time the analyte elutes, which allows more samples to be analyzed per unit of time.

Autosampler

In sequential mode lii

| Acquisition window #1 | Acquisition window #2 | Injection 1 Injection 2 Injection 3

b With column switching a

| Acquisition window #1 | Acquisition window #3 | Injection 1 Injection 3 Injection 5

Analytical column 1

Analytical column 2

I Acquisition window #2 | Acquisition window #4 | Injection 2 Injection 4 Injection 6

Figure 8.3. Column switching or multiplexing using multiple parallel chromatography columns connected to a single mass spectrometer. (a) Schematic of the connections between the autosampler, pumps, HPLC columns, and mass spectrometer for a system containing four parallel HPLC columns. (b) Data acquisition in the conventional sequential mode (a) and data acquisition using a system with two parallel HPLC columns (b).

• UPLC/UHPLC involves particles with a diameter smaller than 2 mm, and it increases chromatographic separation and/or reduces cycle time.

• Sample pooling combined with the use of MRM transitions to selectively monitor the various analytes of interest.

8.7.2 Quantitative In Vivo ADME Studies

LC-MS/MS is used extensively for bioanalysis of drugs in biological matrices such as plasma, blood, urine, and feces from in vivo studies. Triple quadrupole instruments are most powerful for this purpose, and they operate in the MRM mode monitoring the ana-lyte, the internal standard and, if needed, metabolites. To obtain absolute drug levels, a standard curve is prepared and analyzed in the same batch of samples. Separately prepared quality control samples provide information about the ruggedness of the assay. Methods to support drug discovery studies can be developed in about 15 min, but methods to support GLP (good laboratory practice) toxicology and clinical bioanalysis require extensive validation (Vishwanathan et al. 2007; Savoie et al. 2010). To increase the capacity of in vivo pharmacokinetic screening, the following approaches can be deployed:

• Sample pooling: Samples from in vivo studies with different compounds are pooled and all MRM transitions are monitored.

• Cassette or "n-in-one" dosing: A mixture of up to five drugs is administered to the same animals and a triple quadrupole mass spectrometer operating in the MRM mode is used to quantitate all drugs. A risk associated with this technique is that one drug in the mixture may inhibit the metabolism of another drug. This risk can be reduced by reducing the dose. Frequently, a known reference compound is included in the cassette to gauge (albeit crudely) the extent of drug-drug interactions.

• Column switching/multiplexing: As illustrated in Fig. 8.3, column switching or multiplexing can be used to reduce the time spent on monitoring the effluent of a particular chromatography column.

The potential disadvantage of sample pooling and cassette dosing is that complex mixtures - containing multiple drug candidates and many metabolites - are analyzed. Thus, the risk of interference is more pronounced. This risk can be reduced to some extent by avoiding compounds with the same molecular weight or the same molecular weight as a likely metabolite of another compound (e.g., + 16 Da or +32 Da metabolites).

Dried blood spot (DBS) analysis has been used for a long time for sampling blood from newborns to detect metabolic disorders. Recently, DBS analysis has been combined with LC-MS/MS for quantitative bioanalysis (Spooner et al. 2009). This technique involves depositing a small amount of blood (<100 ml) on absorbent paper and allowing it to dry thoroughly. Subsequently, a small circle is punched out of the paper, which is transferred to a vial or 96-well plate for extraction with an organic solvent. The extract is analyzed by LC-MS/MS. The small amount of blood required for DBS analysis enables serial sampling of blood from mice, and it eliminates the need for a parallel PK group in rat toxicology studies. it also facilitates blood sampling from pediat-ric patients, and shipment of samples no longer requires dry ice.

8.7.3 Metabolite Identification

LC-MS/MS is a very powerful tool for metabolite identification. Before introduction of LC-MS/MS, it was hard to obtain structure-specific data for metabolites, and identification was usually based on similarity between the chromatographic retention time of the metabolite and an authentic standard. Although it may not be possible to identify the exact structure of the metabolite via LC-MS/MS, the information may be sufficient for chemists to address specific metabolic liabilities in the next generation of compounds. Later in the discovery stage, metabolite identification via LC-MS/MS can be used to identify the toxicology species with metabolic pathways most similar to humans. These studies are usually performed with liver microsomes, S9 or hepatocytes. it is also conceivable that a metabolite is responsible for part, or all, of the observed efficacy. Metabolic profiling may help resolve this PK-PD (pharmacokinetic-pharmacodynamic) disconnect. in the development stage, a greater understanding of the metabolic fate is desired and detailed LC-MS/MS studies are performed in particular to address concerns associated with the Metabolites in Safety Testing (MIST) guidance (see Chap. 6).

Interpretation of MS/MS spectra is still time-consuming. However, it is made easier by accurate mass measurements, which can help narrow down the number of possible molecular formulas for the metabolites and the assignment of fragment ions in MS/ MS spectra (vide supra). Sometimes, it may be possible to propose the exact site of metabolism (e.g., N-dealkylation), but frequently identification is limited to a Markush structure in which the site of metabolic modification is associated with a part of the molecule. The latter is most common for hydroxylation reactions. To obtain unambiguous information it may be necessary to isolate the metabolite and obtain NMR data. Additional tools available to facilitate metabolite identification are:

• Chemical derivatization

• Hydrogen-deuterium exchange

• Studies with structurally related analogs

Finally, a metabolic profile obtained via LC-MS/MS is qualitative only. The ionization efficiencies can vary widely across metabolites, in particular if a basic center that enhances ionization in the positive ion mode has been eliminated from the drug candidate via metabolism (It is possible to use nanospray to reduce differences in ioniza-tion efficiency, but this technique is not routinely available (Hop et al. 2005)). Quantitative information can be obtained by splitting the flow and acquiring UV data (although this technique is not necessarily completely accurate either). Ideally, the drug is radiolabeled, and the flow is split to count absolute radioactivity.

Mass Defect Filtering

All molecules have a specific mass defect, which reflects the difference between the exact mass and nominal mass of all atoms (see Table 8.6). For example, the exact mass of the [M + H]+ ions of muraglitazar ([C29H29N2O7]+) is 517.1967 Da and the mass defect is 0.1967 Da. Most phase 1 metabolites, in particular mono- or di-hydroxylated metabolites, have a mass defect fairly similar to that of the parent compound. Thus, full-scan high-resolution MS data can be filtered using a specific mass defect window close to that of the parent compound (Zhang et al. 2007, 2009). Note that phase 2 metabolites, such as sulfates or glucuronides, change the mass defect considerably, and, therefore, different filters need to be deployed to facilitate detection of these metabolites.

8.7.4 MALDI Tissue Imaging

Knowing the distribution of drugs in tissues can facilitate explanation of observed efficacy or toxicity. Prior to a human mass balance study, a quantitative whole body autoradiography study is performed in rodents (and in a non-rodent if this animal species more closely resembles human pharmacokinetics) to see if drug-

related material is retained in specific tissues or organs. This study requires radiolabeled material, and it is not possible to distinguish the parent compound from metabolites. The latter two disadvantages can be circumvented with matrix-assisted laser desorption/ ionization imaging (MALDI; Cornett et al. 2008; Khatib-Shahidi et al. 2006). With this technique, a solution containing a UV-absorbent matrix is sprayed on a tissue slice. After evaporation of the solvent, the tissue is transferred to the vacuum of the mass spectrometer and a laser is scanned across the tissue slices in discrete steps. This mass selective detection technique allows detection of both the parent compound and metabolites with a spatial resolution of up to 30 mm.

References

Cornett DS, Frappier SL, Caprioli RM (2008) MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue. Anal Chem 80:5648-5653

Hop CECA, Chen Y, Yu LJ (2005) Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ioniza-tion source. Rapid Commun Mass Spectrom 19:3139-3142 Khatib-Shahidi S, Andersson M, Herman JL et al (2006) Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry. Anal Chem 78:6448-6456 Plumb RS, Potts WB III, Rainville PD et al (2008) Addressing the analytical throughput challenges in ADME screening using ultra-performance liquid chromatography/tandem mass spectrometry methodologies. Rapid Commun Mass Spectrom 22:2139-2152 Savoie N, Garofolo F, Van Amsterdam P et al (2010) 2009 white paper on recent issues in regulated bioanalysis from the 3rd calibration and validation group workshop. Bioanalysis 2:53-68 Spooner N, Lad R, Barfield M (2009) Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method. Anal Chem 81:1557-1563 Vishwanathan CT, Bansal S, Booth B et al (2007) Quantitative bioanalyti-cal methods validation and implementation: best practices for chro-matographic and ligand binding assays. Pharm Res 24:1962-1973 Zhang D, Cheng PT, Zhang H (2007) Mass defect filtering on high resolution LC/MS data as a methodology for detecting metabolites with unpredictable structures: identification of oxazole-ring opened metabolites of mur-aglitazar. Drug Metab Lett 1:287-292 Zhang H, Zhang D, Ray K et al (2009) Mass defect filter technique and its application to drug metabolite identification by high resolution mass spectrometry. J Mass Spectrom 44:999-1016

Additional Readings

Chowdhury SK (2005) Identification and quantification of drugs, metabolites and metabolizing enzymes by LC-MS. Elsevier, Amsterdam, The Netherlands

Hop CECA (2006) LC-MS in drug disposition and metabolism. In: Caprioli RM (ed) The encyclopedia of mass spectrometry, vol 3. The Netherlands, Elsevier, Amsterdam, pp 233-274 Korfmacher WA (2010) Using mass spectrometry for drug metabolism studies, 2nd edn. CRC Press, Boca Raton, FL Ramanathan R (2009) Mass spectrometry in drug metabolism and pharmacokinetics. Wiley, New York

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